华支睾吸虫lncRNA EV19对人肝星状细胞纤维化相关基因的调控研究  

Clonorchis sinensis lncRNA EV19 regulates genes related to fibrosis in human hepatic stellate cells

作　　者：王晶[1] 唐斌[1] 王兴洋 司光泉 刘志娜 丁静 刘明远[1] 刘晓雷[1] 王洋[1] WANG Jing;TANG Bin;WANG Xingyang;SI Guangquan;LIU Zhina;DING Jing;LIU Mingyuan;LIU Xiaolei;WANG Yang(State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases/College of Veterinary Medicine,Jilin University,Changchun 130062,China)

机构地区：[1]吉林大学、动物医学学院、人畜共患传染病重症诊治全国重点实验室,吉林长春130062

出　　处：《中国兽医科学》2024年第6期718-727,共10页Chinese Veterinary Science

基　　金：中央高校基本科研业务费专项(JLUXKJC2021ZZ10)。

摘　　要：为探究华支睾吸虫长链非编码RNA(lncRNA),对人肝星状细胞(LX-2细胞)纤维化相关因子的影响,通过生物信息学分析初步筛选出8种可能与纤维化相关的华支睾吸虫lncRNA,进一步利用RT-qPCR证明其中lncRNA EV1、EV17、EV19在华支睾吸虫成虫及其胞外囊泡中均表达。随后构建pcDNA3.1-lncRNA EV19、pcDNA3.1-lncRNA EV17、pcDNA3.1-lncRNA EV1过表达载体,转染LX-2细胞;收集转染后的细胞,通过RT-q PCR和Western-blot分别检测α-SMA、Col1、Col3基因的转录和蛋白表达水平;并利用CCK-8检测lncRNA对LX-2细胞增殖的影响。最后,利用RT-qPCR及双荧光素酶报告基因试验验证lncRNA EV19作用靶标。结果显示,与对照组相比,lncRNAEV19过表达组中α-SMA、Col1、Col3在转录水平和蛋白水平均显著升高(P<0.05),且能促进LX-2细胞的增殖。RT-qPCR结果显示,过表达lncRNA EV19的LX-2细胞中miR-19b-1-5p、miR-29b-1-5p、miR-940表达量降低;双荧光素酶报告基因试验证实lncRNA EV19靶向miR-19b-1-5p。以上结果表明,华支睾吸虫lncRNA EV19可能通过负调控miR-19b-1-5p促进LX-2细胞的增殖,进而引起纤维化的发生。In order to investigate the impact of Clonorchis sinensis long non-coding RNAs(lncRNAs)on human hepatic stellate cells(LX-2 cells)fibrosis-related factors,eight lnc RNA of C.sinensis possibly related to fibrosis were initially screened through bioinformatics analysis.And RT-qPCR confirmed the expression of the lnc RNAs EV1,EV17,and EV19 in adult C.sinensis and their extracellular vesicles.Then,pc DNA3.1-lnc RNA EV19,pc DNA3.1-lnc RNA EV17,and pc DNA3.1-lnc RNA EV1 overexpression vectors were constructed,and transfected into LX-2 cells.The transfected cells were collected,and the levels of transcription and protein expression of theα-SMA,Col 1,and Col 3 genes were detected using RT-q PCR and Western-blot,respectively.Additionally,the CCK-8 assay was used to detect the influence of lnc RNA on the proliferation of LX-2 cells.Finally,RT-q PCR and a double luciferase reporter assay were performed to verify the target of lnc RNA EV19.The results showed that,compared with the control group,the overexpression of lnc RNA EV19 significantly increased the transcription and protein levels ofα-SMA,Col 1,and Col 3(P<0.05),and promoted the proliferation of LX-2 cells.The RT-q PCR results showed that the expression levels of miR-19b-1-5p,miR-29b-1-5p,and miR-940 were decreased in LX-2 cells overexpressing lnc RNA EV19.Dual-luciferase reporter assay confirmed that lnc RNA EV19 targets miR-19b-1-5p.These results indicate that the lnc RNA EV19 from C.sinensis may promote the proliferation of LX-2cells and induce fibrosis by negatively regulating miR-19b-1-5p.

关 键 词：华支睾吸虫 人肝星状细胞 肝纤维化 长链非编码RNA micro RNA 

分 类 号：S852.735[农业科学—基础兽医学]